This grant has funded work on altered peptide ligands (APL) for therapy of autoimmune disease, specifically multiple sclerosis (MS). Work undertaken during the previous five years has provided a framework for the first clinical trials on patients with relapsing remitting MS. In the next grant period we propose to carry out pre-clinical experiments that will optimize APL therapy in animal models of MS, and to develop the next generation of APL therapy utilizing techniques involving vaccination with naked DNA. We will also demonstrate how microbes have the capacity to modulate autoimmune disease via structural mimicry with self-molecules. The specific aims of this grant are to 1) subvert epitope spreading with APL. In this aim we will see whether after intermolecular epitope spreading has occurred, if an APL which targets a single epitope on a single myelin molecule, would be able to suppress ongoing EAE, and reverse paralysis. This is a stringent demand, but will be a useful paradigm to optimize therapeutic regimens in Phase II clinical trials with relapsing remitting MS patients. In Aim 2 we will analyse how APL pulsed dendritic cells will serve to reverse EAE. In Aim 3 we will attempt APL therapy via vaccination with DNA minigenes encoding myelin epitopes. We have succeeded in preventing EAE with DNA vaccination using a minigene encoding a myelin PLP epitope. We will optimize this approach utilizing myelin minigenes paired in tandem with cytokine and chemokine constructs. Finally in Aim 4 we will study APL as microbial mimics that modulate EAE. We have succeeded with preliminary studies, in demonstrating subversion of epitope spreading, with microbial sequences that mimic myelin epitopes. The optimization of such APL derived from microbes will be studied here. This work with APL may be applicable not only to MS, but to other autoimmune diseases including juvenile diabetes, rheumatoid arthritis, and inflammatory bowel disease.